Cyanobacteria are small, prokaryotic, generally aquatic organisms that can be genetically manipulated to be capable of utilizing light and CO2 to produce compounds of interest, such as biofuels, industrial chemicals, pharmaceuticals, nutrients, carotenoids, food supplements, etc. Because cyanobacterial cells are capable of fixing carbon dioxide as a carbon source for autotrophic growth, they do not require the costly input of organic carbon as a starting material. Further, the CO2 that is utilized by the cyanobacterial culture can be derived from any source, such as a waste byproduct of industrial production. In this way, Cyanobacteria can be used to recycle CO2 to desired products, such as biofuel.
Various cyanobacterial species have been genetically enhanced to produce compounds of interest. The transformation of the cyanobacterial genus Synechococcus with genes that encode enzymes that can produce ethanol for biofuel production has been described (U.S. Pat. Nos. 6,699,696 and 6,306,639, both to Woods et al,). The transformation of the cyanobacterial genus Synechocystis has been described, for example, in PCT/EP2009/000892 and in PCT/EP2009/060526.
The cyanobacteria as a whole are a very divergent group of organisms. Due to this diversity, it is often difficult to find a method to effectively and efficiently transform a given host cyanobacterial species. Further, it is also often difficult for the inserted DNA vehicle to replicate adequately once it is present in the host cyanobacterial cell.
Certain strains of cyanobacteria can be naturally and relatively easily transformed. Other cyanobacterial strains can be transformed, for example, by the use of conjugation or electroporation. Some cyanobacterial strains are difficult to transform by any known means. For many of these types of difficult to transform strains, specific methods of preparing the cells for transformation, as well as specific methods of allowing entry of the foreign DNA into the cells, need to be designed.
The transfer of foreign genes into cyanobacteria often involves the construction of vectors having a backbone from a broad-host range bacterial plasmid, such as RSF1010. The RSF1010-based vector has been widely used as a conjugation vector for transforming bacteria, including cyanobacteria (Mermet-Bouvier et al. (1993) “Transfer and replication of RSF1010-derived plasmids in several cyanobacteria of the genera Synechocystis and Synechococcus” Current Microbiology 27:323-327). This plasmid has an E. coli origin of replication, but does not have a cyanobacterial origin of replication.
Several endogenous plasmids from Synechococcus sp. PCC7002 have been utilized as a backbone plasmid to prepare vectors for heterologous gene expression (Xu et al., Photosynthesis Research Protocols 684:273-293 (2011). Other vectors for transformation of cyanobacteria include the pDUI-based vectors, The pDUI origin of replication is best suited for filamentous cyanobacteria, however. Attempts to transform certain species of cyanobacteria, such as Cyanobacterium sp. ABICyanol, with either RSF1010 or pDUI-based shuttle vectors were previously unsuccessful.
The cyanobacterial genus Cyanobacterium was first established in 1983 (see Rippka et al. (2001), Bergey's Manual of Systematic Bacteriology, Vol. 1, p, 497-498). In general, the genus differs from the genus Synechococcus by differences in DNA base composition and by differences in sensitivity to cyanophages (Moro, et al., 2007, Algological Studies, 123:1-15). Members of the Cyanobacterium genus are often found in thermal mats.
The species Cyanobacterium ABICyanol is a coccoid, unicellular cell somewhat similar to Synechococcus when viewed under the microscope. Cells of Cyanobacterium ABICyanol appear to have a substantial layer of mucilaginous sheath covering each individual cell. This mucilage can participate in the formation of cellular aggregates or “clumps”. The species differs from other species in the Cyanobacterium genus, as well as from other cyanobacteria such as Synechococcus and Synechocystis, by differences in the carotenoid and chlorophyll composition. The species also appears to differ from other cyanobacteria, such as the above two species, by differences in its 16S rDNA and its internal transcribed spacer rDNA (ITS) composition.
What is needed in the art is a new cyanobacterial strain that grows relatively quickly, is tolerant to various environmental stresses, and can successfully harbor foreign genes for the production of compounds of interest, such as biofuels.